Disclosed are means of inducing and accelerating neurological recovery subsequent to a stroke through administration of amniotic fluid derived stem cells. In one embodiment stem cells are isolated from amniotic fluid and expanded under conditions allowing for expression of SSEA3, SSEA4, Tra1-60, Tra1-81, Tra2-54, Oct-4 and CD105. Said cells are subsequently administered into a patient having undergone a stroke, so as to induce direct regeneration (through transdifferentiation and replacement of neural tissue), as well as indirect regeneration (through production of growth factors that augment endogenous regenerative mechanisms while inhibiting degenerative mechanisms). In some embodiments factors produced by said amniotic fluid stem cells may be utilized instead of cells themselves. Said factors may include proteins, peptides, conditioned media, exosomes, or microvesicles.
Legal claims defining the scope of protection, as filed with the USPTO.
. A method of accelerating recovery subsequent to a brain injury associated with a loss of oxygen perfusion comprising the steps of:
. The method of, wherein said amniotic fluid is obtained from amniocentesis.
. The method of, wherein said amniotic fluid is obtained from amniotic membranes.
. The method of, wherein said loss of oxygen perfusion is caused by a stroke.
. The method of, wherein said stroke is an ischemic stroke.
. The method of, wherein said stroke is a hemorrhagic stroke.
. The method of, wherein said stroke is a transient ischemic attack.
. The method of, wherein said loss of oxygen perfusion is a traumatic brain injury.
. The method of, wherein said traumatic brain injury is chronic traumatic encephalopathy.
. The method of, wherein said loss of oxygen perfusion is caused by vascular disease of the brain.
. The method of, wherein said loss of oxygen perfusion is result of global cerebral ischemia.
. The method of, wherein said global cerebral ischemia occurs as a neonate.
. The method of, wherein said neonatal global cerebral ischemia results in cerebral palsy.
. The method of, wherein said global cerebral ischemic occurs due to asphyxiation.
. The method of, wherein said global cerebral ischemic occurs due to drowning.
. The method of, wherein said global cerebral ischemic occurs due to a cardiac event.
. The method of, wherein said amniotic cells with regenerative activity possess expression of the markers SSEA3, SSEA4, Tra1-60, Tra1-81, Tra2-54, Oct-4 and CD105.
Complete technical specification and implementation details from the patent document.
This application claims the benefit of priority to U.S. Provisional Application No. 62/393,608, filed Sep. 12, 2016, which is incorporated herein by reference in its entirety
The invention pertains to the field of tissue regeneration, more specifically, the invention pertains to regeneration of neural tissue after an ischemic insult, more specifically, the invention teaches the use of amniotic fluid derived cells in the induction and acceleration of post-stroke recovery.
Various insults to the brain of ischemic nature are associated with an initial loss of tissue due to immediate death, however a “stunned” are of neural tissue is susceptible to recovery. This temporarily dysfunctional tissue is termed the “penumbra”. Furthermore areas of the brain have been demonstrated to possess regenerative activity after insult, however factors secreted by the insult prevent regeneration [1]. The pathogenesis of ischemic induced neurocognitive deficits involves apoptosis of neuroproliferative cells in the subgranular zone of the hippocampus, a region in the brain vital for learning and memory [2, 3]. Several studies have demonstrated a steep, long-term decline in subgranular neurogenesis in the dentate gyrus following ischemic damage [4] and direct irradiation of the hippocampus has been shown to result in pronounced cognitive deficits [5]. The cognitive deficits following various types of ischemia include deficits of learning, memory, and ability for spatial processing [6]. To date, no medication has been approved for induction and/or acceleration of post-ischemic brain insult recovery.
Various aspects of the invention relating to the above are enumerated in the following paragraphs:
Aspect 1: A method of accelerating recovery subsequent to a brain injury associated with a loss of oxygen perfusion comprising the steps of: a) obtaining amniotic fluid; b) extracting from said amniotic fluid a population of cells with regenerative activity; c) expanding said amniotic fluid cells with regenerative activity in a manner to allow for increased number of cells while maintaining said regenerative activity; d) administering said expanded amniotic fluid derived cells with regenerative activity into a patient in need of treatment.
Aspect 2: The method of aspect 1, wherein said amniotic fluid is obtained from amniocentesis.
Aspect 3: The method of aspect 1, wherein said amniotic fluid is obtained from amniotic membranes.
Aspect 4: The method of aspect 1, wherein said loss of oxygen perfusion is caused by a stroke.
Aspect 5: The method of aspect 4, wherein said stroke is an ischemic stroke.
Aspect 6: The method of aspect 4, wherein said stroke is a hemorrhagic stroke.
Aspect 7: The method of aspect 4, wherein said stroke is a transient ischemic attack.
Aspect 8: The method of aspect 1, wherein said loss of oxygen perfusion is a traumatic brain injury.
Aspect 9: The method of aspect 8, wherein said traumatic brain injury is chronic traumatic encephalopathy.
Aspect 10: The method of aspect 1, wherein said loss of oxygen perfusion is caused by vascular disease of the brain.
Aspect 11: The method of aspect 1, wherein said loss of oxygen perfusion is result of global cerebral ischemia.
Aspect 12: The method of aspect 11, wherein said global cerebral ischemia occurs as a neonate.
Aspect 13: The method of aspect 12, wherein said neonatal global cerebral ischemia results in cerebral palsy.
Aspect 14: The method of aspect 12, wherein said global cerebral ischemic occurs due to asphyxiation.
Aspect 15: The method of aspect 12, wherein said global cerebral ischemic occurs due to drowning.
Aspect 16: The method of aspect 12, wherein said global cerebral ischemic occurs due to a cardiac event.
Aspect 17: The method of aspect 1, wherein said amniotic cells with regenerative activity are amniotic fluid stem cells.
Aspect 18: The method of aspect 1,wherein said amniotic cells with regenerative activity are amniotic fluid mesenchymal stem cells.
Aspect 19: The method of aspect 1, wherein said amniotic cells with regenerative activity possess an epitheliod morphology.
Aspect 20: The method of aspect 1, wherein said amniotic cells with regenerative activity possess expression of the markers SSEA3, SSEA4, Tra1-60, Tra1-81, Tra2-54, Oct-4 and CD105.
Aspect 21: The method of aspect 20, wherein said amniotic fluid cells are capable of differentiating into bone, cartilage and adipose tissue.
Aspect 22: The method of aspect 20, wherein said amniotic fluid cells possess less than 5% expression of SSEA1.
Aspect 23: The method of aspect 20, wherein said amniotic fluid cells are characterized by senescence after about 60 population doubling.
Aspect 24: The method of aspect 20, wherein said amniotic fluid cells are characterized by senescence after about 300 population doubling.
Aspect 25: The method of aspect 20, wherein said amniotic fluid cells are derived from a mammal.
Aspect 26: The method of aspect 20, wherein said mammal possesses a hemochorial placenta.
Aspect 27: The method of aspect 20, wherein said mammal is a human.
Aspect 28: The method of aspect 1, wherein said amniotic fluid is extracted in the first trimester.
Aspect 29: The method of aspect 1, wherein said amniotic fluid is extracted in the second trimester.
Aspect 30: The method of aspect 1, wherein said amniotic fluid is extracted in the third trimester.
Aspect 31: The method of aspect 1, wherein said amniotic fluid cells express one or more markers selected from a group comprising of: HLA class I, CD13, CD44, and CD49b.
Aspect 32: The method of aspect 1, wherein said amniotic fluid cells with regenerative activity are generated by the steps comprising of: a) harvesting amniotic fluid; b) centrifuging the amniotic fluid; c) plating cells onto plates coated with fibronectin in medium with 2% serum; d) selecting colonies which adhere to the plates; and e) isolating mortal, epithelioid morphology cells.
Aspect 33: The method of aspect 1, wherein said amniotic fluid derived cells with regenerative potential possess ability to inhibit secretion of inflammatory cytokines.
Aspect 34: The method of aspect 33, wherein said inflammatory cytokines are selected from a group comprising of; a) IFN-gamma; b) TNF-alpha; c) IL-2; d) IL-7; e) IL-12; f) IL-15; g) IL-17; h) IL-18; i) IL-21; j) IL-23; k) IL-27; l) IL-33; m) HMGB-1; and n) TRAIL.
Aspect 35: The method of aspect 1, wherein said amniotic fluid derived cells with regenerative potential are engineered for enhanced vivo persistence through transfection of a therapeutic peptide sequence encoding an anti-apoptotic gene.
Aspect 36: The method of aspect 35, wherein said enhancement of in vivo persistence is accomplished by enhancing the anti-thrombogenic properties of said amniotic fluid cells.
Aspect 37: The method of aspect 36, wherein said anti-thrombogenic properties are enhanced by inhibiting expression or function of thrombogenic molecules on said amniotic fluid cells.
Aspect 38: The method of aspect 36, wherein said amniotic fluid stem cells are engineered to express anti-thrombin III.
Aspect 39: The method of aspect 35, wherein said enhanced in vivo persistence is accomplished by modifying said amniotic fluid cells to avoid complement mediated lysis.
Aspect 40: The method of aspect 35, wherein said enhanced in vivo persistence is accomplished by modifying said amniotic fluid cells to avoid immune system killing.
Aspect 41: The method of aspect 39, wherein avoidance of complement mediated lysis is accomplished by expression of Decay Accelerating Factor (DAF) protein.
Aspect 42: The method of aspect 40, wherein said avoidance of immune system killing is accomplished by transfection with a molecule that blocks expression of HLA I and/or HLA II.
Aspect 43: The method of aspect 42, wherein said molecule is a siRNA selectively targeting transcripts associated with HLA I and/or HLA II.
Aspect 44: The method of aspect 42, wherein said molecule is a shRNA selectively targeting transcripts associated with HLA I and/or HLA II.
Aspect 45. The method of aspect 42, wherein said molecule is a ribozyme selectively targeting transcripts associated with HLA I and/or HLA II.
Aspect 46: The method of aspect 42, wherein said molecule is an antisense oligonucleotide selectively targeting transcripts associated with HLA I and/or HLA II.
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June 2, 2026
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